It is well established that growth factors play a crucial role in the establishment and maintenance of the transformed phenotype. Evidence is rapidly accumulating that growth factor receptors also play a crucial role in the establishment and maintenance of transformed phenotypes.
The IGF-1R belongs to the family of tyrosine kinase growth factor receptors (Ullrich, et al., Cell, 1990, 61, 203), and is 70% homologous to the insulin growth factor I receptor (Ullrich, et al., EMBO J., 1986, 5, 503). The IGF-1R activated by its ligands (IGF-1, IGF-2 and insulin at supraphysiological concentrations) has been known to be mitogenic in cell cultures. However, in growth-regulated cells, like 3T3 cells and human diploid fibroblasts, IGF-1, by itself, cannot sustain growth of cells in serum-free medium (SFM), but requires the cooperation of other growth factors, for instance PDGF and/or EGF, which, by themselves, also fail to induce a mitogenic response. Scher, et al., Biochem. Biophys. Acta, 1979, 560, 217; and Stiles, et al., Proc. Natl. Acad. Sci. U.S.A., 1979, 76, 1279.
Recently, the importance of the IGF-1R in cell growth has been confirmed in vivo by the finding that mouse embryos with a targeted disruption of the IGF-1R and IGF-2 genes have a size at birth that is only 30% the size of wild type littermates. Liu, et al., Cell, 1993, 75, 59; and Baker, et al., Cell, 1993, 73, 73. 3T3-like cells derived from these mouse embryos devoid of IGF-1Rs (R.sup.- cells) do not grow at all in SFM supplemented by a variety of growth factors, which can sustain the growth of cells derived from wild type littermate embryos (W cells) and other 3T3-cells. Sell, et al., Mol. Cell. Biol., 1994, 14, 3604. R.sup.- cells grow in 10% FBS at a rate that is roughly 40% the rate of W cells, with all phases of the cell cycle being equally elongated. Sell, et al., Mol. Cell. Biol., 1994, 14, 3604. R.sup.- cells are also refractory to transformation by SV40 large T antigen, by an activated ras or a combination of both (Sell, et al., Proc. Natl. Acad. Sci. U.S.A., 1993, 90, 11217; and Sell, et al., Mol. Cell. Biol., 1994, 14, 3604), or by overexpressed growth factor receptors, such as the EGF receptor (Coppola, et al., Mol. Cell. Biol., 1994, 14, 4588), the PDGF .beta. receptor (DeAngelis, et al., J. Cell. Physiol., 1995, 164, 214) and the insulin receptor (Miura, et al., Cancer Res., 1995, 55, 663), all conditions that readily transform cells from wild type littermate embryos or other 3T3-like cells with a physiological number of IGF-1Rs. Conversely, overexpression and/or constitutive activation of IGF-1R in a variety of cell types leads to ligand-dependent growth in SFM and to the establishment of a transformed phenotype. Kaleko, et al., Mol. Cell. Biol., 1990, 10, 464; McCubrey, et al., Blood, 1991, 78, 921; Pietrzkowski, et al., Mol. Cell. Biol., 1992, 12, 3883; Liu, et al., Cell, 1993, 75, 59; Sell, et al., Mol. Cell. Biol., 1994, 14, 3604; Coppola, et al., Mol. Cell. Biol., 1994, 14, 4588; and Surmacz, et al., Exp. Cell Res., 1995, 218, 370.
The importance of the IGF-1 receptor in the control of cell proliferation is also supported by the observation that many cell types in culture are stimulated to grow by IGF-I (Goldring, et al., Crit. Rev. Eukaryot. Gene Expr., 1991, 1, 301; and Baserga, et al., Crit. Rev. Eukaryot. Gene Expr., 1993, 3, 47), and these cell types include human diploid fibroblasts, epithelial cells, smooth muscle cells, T lymphocytes, myeloid cells, chondrocytes, osteoblasts as well as the stem cells of the bone marrow. Using antisense expression vectors or antisense oligonucleotides to the IGF-1 receptor RNA, it has been shown that interference with IGF-1 receptor leads to inhibition of cell growth. The antisense strategy was successful in inhibiting cellular proliferation in several normal cell types and in human tumor cell lines. Baserga, Cell, 1994, 79, 927. Growth can also be inhibited using peptide analogues of IGF-1 (Pietrzkowski, et al., Cell Growth & Diff., 1992, 3, 199; and Pietrzkowski, et al., Mol. Cell. Biol., 1992, 12, 3883), or a vector expressing an antisense RNA to the IGF-1 RNA (Trojan, et al., Science, 1993, 259, 94). The IGF autocrine or paracrine loop is also involved in the growth promoting effect of other growth factors, hormones (for instance, growth hormone and estrogens), and oncogenes like SV40 T antigen and c-myb, and in tumor suppression, as in the case of WT1 (Baserga, Cell, 1994, 79, 927).
The important role of IGF-1R in the establishment and maintenance of the transformed phenotype is supported by other findings. Antisense oligonucleotides or antisense expression plasmids against either IGF-2 (Christophori, et al., Nature, 1994, 369, 414; and Rogler, et al., J. Biol. Chem., 1994, 269, 13779), IGF-1 (Trojan, et al., Proc. Natl. Acad. Sci. U.S.A., 1992, 89, 4874; and Trojan, et al., Science, 1993, 259, 94) or the IGF-1R (Sell, et al., Proc. Natl. Acad. Sci. U.S.A., 1993, 90, 11217; Baserga, Cell, 1994, 79, 927; Resnicoff, et al., Cancer Res., 1994, 54, 2218; Resnicoff, et al., Cancer Res., 1994, 54, 4848; and Shapiro, et al., J. Clin. Invest., 1994, 94, 1235), antibodies to the IGF-1R (Arteaga, et al., Breast Canc. Res. Treatm., 1992, 22, 101; and Kalebic, et al., Cancer Res., 1994, 54, 5531), and dominant negative mutants of the IGF-1R (Prager, et al., Proc. Natl. Acad. Sci. U.S.A., 1994, 91, 2181; and Li, et al., J. Biol. Chem., 1994, 269, 32558), can all reverse the transformed phenotype, inhibit tumorigenesis, and induce loss of the metastatic phenotype (Long, et al., Cancer Res., 1995, 54, 1006). An overexpressed IGF-1R has been found to protect tumor cells in vitro from etoposide-induced apoptosis (Sell, et al., Cancer Res., 1995, 55, 303) and, even more dramatically, that a decrease in IGF-1R levels below wild type levels caused massive apoptosis of tumor cells in vivo (Resnicoff, et al., Cancer Res., 1995, 55, 2463).
Expression of an antisense RNA to the IGF-1R RNA in C6 rat glioblastoma cells not only abrogates tumorigenesis in syngeneic rats, but also causes complete regression of established wild type tumors. Resnicoff, et al., Cancer Res., 1994, 54, 2218; and Resnicoff, et al., Cancer Res., 1994, 54, 4848. Cells expressing an antisense RNA to the IGF-1R RNA or cells pre-incubated with antisense oligonucleotides to the IGF-1R RNA completely lose their tumorigenicity when injected in either syngeneic or nude mice. Resnicoff, et al., Cancer Res., 1994, 54, 2218; and Resnicoff, et al., Cancer Res., 1994, 54, 4848. The injected cells were suspected of undergoing apoptosis or some form of cell death. Dying cells, however, are very difficult to demonstrate, because dying cells, especially in vivo, disappear very rapidly, and one is left with nothing to examine.
Tumors and other neoplastic tissues are known to undergo apoptosis spontaneously or in response to treatment. Examples include several types of leukemia, non-Hodgkin's lymphoma, prostate tumor, pancreatic cancer, basal and squamous cell carcinoma, mammary tumor, breast cancer, and fat pad sarcoma. Several anticancer drugs have been shown to induce apoptosis in target cells. Buttyan, et al., Mol. Cell. Biol., 1989, 9, 3473; Kaufmann, Cancer Res., 1989, 49, 5870; and Barry, et al., Biochem. Pharmacol., 1990, 40, 2353. Certain mildly adverse conditions can result in the injured cell dying by programmed cell death, including hyperthermia, hypothermia, ischemia, and exposure to irradiation, toxins, and chemicals. It should be noted that many of these treatments will also result in necrosis at higher doses, suggesting that mild injury to a cell might induce a cell to commit suicide, perhaps to prevent the inheritance of a mutation, while exposure to severe conditions leads directly to cell death by necrosis.
Apoptosis refers to cell death, including, but not limited to, regression of primary and metastatic tumors. Apoptosis is a programmed cell death which is a widespread phenomenon that plays a crucial role in the myriad of physiological and pathological processes. There exists a homeostatic control of cell number thought to result from the dynamic balance between cell proliferation and cell death. In contrast, necrosis refers to an accidental cell death which is the cell's response to a variety of harmful conditions and toxic substances.
Apoptosis, morphologically distinct from necrosis, is a spontaneous form of cell death that occurs in many different tissues under various conditions. This type of cell death typically occurs in scattered cells and progresses so rapidly that it is difficult to observe.
The cell death process of apoptosis occurs in two stages. The cell undergoes nuclear and cytoplasmic condensation, eventually breaking into a number of membrane-bound fragments containing structurally intact apoptotic bodies, which are phagocytosed by neighboring cells and rapidly degraded. Apoptosis is observed in many different tissues, healthy and neoplastic, adult and embryonic. Death occurs spontaneously, or is induced by physiological or noxious agents. Apoptosis is a basic physiological process that plays a major role in the regulation of cell populations.
The death process is difficult to observe due to the rapidity of the process and the reduced amount of inflammation. For these reasons, quantification of apoptosis is often difficult. A method of measuring the duration of the histologically visible stages of apoptosis (3 hours in normal rat liver) and present a formula by which to calculate the cell loss rate by apoptosis. Bursch, et al., Carcinogenesis, 1990, 11, 847.
Nonetheless, testing agents such as growth factors and growth factor receptors for their ability to maintain or suppress transformed phenotypes remains difficult. In order to obtain an accurate account of the tumor suppressive ability, testing should be performed in vivo. Therapies such as direct injection or implantation of toxic treatments, tissue samples, and chemotherapy often jeopardizes the overall health of the patient. The present invention provides a method of inducing resistance to tumor growth with markedly reduced side effects to the patient.